Different Oligomeric States of the Tumor Suppressor p53 Show Identical Binding Behavior towards the S100β Homodimer

Abstract The tumor suppressor protein p53 is a transcription factor that is referred to as the “guardian of the genome” and plays an important role in cancer development. p53 is active as a homotetramer; the S100β homodimer binds to the intrinsically disordered C‐terminus of p53 affecting its transcriptional activity. The p53/S100β complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100β exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100β complexes by ESI‐MS, XL‐MS, and SPR. Wild‐type p53 and single amino acid variants, representing different oligomeric states of p53 were individually investigated regarding their binding behavior towards S100β. The stoichiometry of the different p53/S100β complexes were determined by ESI‐MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100β dimer. In addition, XL‐MS revealed the topologies of the p53/S100β complexes to be independent of p53’s oligomeric state. With SPR, the thermodynamic parameters were determined for S100β binding to tetrameric, dimeric, or monomeric p53 variants. Our data prove that the S100β homodimer binds to different oligomeric states of p53 with similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100β interaction.


Protein Expression and Purification
Expression and purification of full-length human p53 (wild-type, L344A, L344P) were performed as previously described [1] .
All chemicals were obtained from Sigma at the highest purity if not stated otherwise. Milli-Q-water was used for all reactions.

Covalent Modification of Proteins
N-hydroxysuccinimide (NHS) esters, such as disuccinimidyl dibutyric urea (DSBU, CF Plus Chemicals), mainly react with the primary amines of lysines and the N-termini of proteins. Reactions with amino acids that contain hydroxy groups, such as serines, threonines, and tyrosines, are also observed. A recent comparative community-wide cross-linking mass spectrometry (XL-MS) study [3] reported that serines, threonines, and tyrosines account for ~30% of NHS ester reaction sites. In human p53, here are 21 primary amine groups and 69 hydroxy groups there are 90 potential reactive sites of p53. The same calculation for S100β with 8 primary amine groups and 9 hydroxy groups results in 17 potential reactive sites. Conclusively, the molar concentration of nucleophilic residues in solution is [p53] x p53reactive sites + [S100β] x S100β reactive sites = 6 µM x 42 + 6 µM x 11 = 318 µM. Thus, the molar ratio of DSBU over the nucleophilic residues in solution is 120 µM / 318 µM = 0.38, corresponding to ~ 0.4 equivalents of the cross-linker DSBU.
All covalent modification experiments were performed at 4°C in buffer (50 mM HEPES, 300 mM NaCl, 2.5 mM TCEP, and 10% glycerol, pH 7.2). Each variant of p53 (wild-type, L344A, and L344P) was used at a final concentration of 6 μM, S100β was used at a final concentration of 6 μM, and DSBU with a final concentration of 0.12 mM (20-fold molar excess of p53). 1 mM of calcium chloride was added to calcium-containing DSBU-modified samples, while for calcium-depleted DSBU-modified samples 1 mM of the chelating agent EGTA was added. Quenching of cross-linking reactions was performed by adding 20 mM of ammonium bicarbonate after 60 minutes. Buffer exchange was conducted immediately after the addition of ammonium bicarbonate.

Buffer Exchange
Prior to ESI-MS experiments, buffer exchange to 500 mM ammonium acetate (pH 6.8) was performed. Buffer exchange of the cross-linked samples (L344P and L344A variants of p53) was performed with Amicon Ultra centrifugal filter units (molecular weight cut-off 30 kDa, Merck Millipore). For the cross-linked samples of wild-type p53, an online buffer exchange (OBE) procedure was performed. The sample was loaded via the autosampler on the Ultimate 3000 RSLC nano-HLPC system (Thermo Fischer Scientific). The sample was then desalted and buffer exchanged with a selfpacked polyacrylamide P6 column (BioRad).

Cross-linking of Proteins
All cross-linking reactions were performed in three replicates and analyzed separately.

DSBU cross-linking
A solution containing 6 μM wild-type p53 or L344A or L344P variants of p53 and 6 μM S100β in buffer (50 mM HEPES, 1 mM CaCl2, 300 mM NaCl, and 2.5 mM TCEP, pH 7.2) was cross-linked at 4°C for 1 h with DSBU at 100-fold molar excess. DSBU was dissolved in neat dimethyl sulfoxide (DMSO) at a concentration of 0.6 mM, immediately before adding it to the protein solution. The reaction was quenched by adding ammonium bicarbonate to a final concentration of 20 mM.

Sulfo-SDA crosslinking
A solution containing 6 μM wild-type p53 or L344A or L344P variants of p53 and 6 μM S100β in buffer (50 mM HEPES, 1 mM CaCl2, 300 mM NaCl, and 2.5 mM TCEP, pH 7.2) was cross-linked at 4°C for 2 hrs with EDC and sulfo-NHS at 500-fold molar excess. Sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA) was dissolved in DMSO at a concentration of 0.6 mM, immediately before adding it to the protein solution. After the reaction with EDS/sulfo-NHS the sample was irradiated for 15 s at 365 nm (Panasonic Aicure UJ30/35 in a home-built irradiation chamber. The reaction was quenched by adding ammonium bicarbonate to a final concentration of 20 mM.

Enzymatic Digestion
All cross-linked samples were reduced to a volume of 2-3 µl in a SpeedVac concentrator. 25 µl of 8 M urea in 400 mM ammonium bicarbonate was added to each sample. After 5 min of sonication, samples were incubated with dithiothreitol (DTT, 6.4 mM) for 30 min at 56°C and iodoacetamide (12.5 mM) for 20 min at room temperature in the dark. The alkylation reaction was quenched by adding 9 mM of DTT. After diluting the samples to 1 M urea with water, they were incubated overnight at 37°C with AspN (NEB) at an 1:20 (w/w) enzyme-substrate ratio and digested with trypsin (Promega) at 1:20 (w/w) for 4 h at 37 °C. All proteolytic digestions were stopped by adding 10% (v/v) TFA.
For the settings of the timsTOF Pro mass spectrometer, the following parameters were adapted, starting with the parallel accumulation serial fragmentation (PASEF) method for standard proteomics. The values for mobility-dependent collision energy ramping were set to 95 eV at an inversed reduced mobility (1/k0) of 1.6 V s/cm 2 and 23 eV at 0.73 V s/cm 2 . Collision energies were linearly interpolated between these two 1/k0 values and kept constant above or below. No merging of TIMS scans was performed. Target intensity per individual PASEF precursor was set to 20 000. The scan range was set between 0.6 and 1.6 V s/cm 2 with a ramp time of 166 ms. 14 PASEF MS/MS scans were triggered per cycle (2.57 s) with a maximum of seven precursors per mobilogram. Precursor ions in an m/z range between 100 and 1700 with charge states ≥2+ and ≤8+ were selected for fragmentation. Active exclusion was enabled for 0.4 min (mass width 0.015 Th, 1/k0 width 0.015 V s/cm 2 ).
MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the project accession PXD029914, username: reviewer_pxd030001@ebi.ac.uk; password: 0JIKK9rp.
Mapping of cross-links was visualized with xiNET [4] .

Surface Plasmon Resonance (SPR)
All SPR measurements were acquired with MP-SPR Navi 200 OTSO instrument (BioNavis). A CMDP sensorchip (Xantac) was used to perform experiments to investigate the protein-protein binding interactions between p53 (tetrameric wild-type, dimeric variant L344A, monomeric variant L344P) and S100β. All buffers were degassed and filtered prior to SPR experiments. Pre-concentration experiments were performed in order to test the optimal immobilization pH (3.6, 3.9, 4.2) of 10 mM sodium acetate, where a pH value of 3.9 was found to be the most optimal.
The running buffer for ligand immobilization and binding experiments consisted of 50 mM HEPES, 300 mM NaCl, 2.5 mM TCEP, and 1 mM of CaCl2 (pH 7.2). For protein immobilization, 5 μM of S100β was diluted with 10 mM sodium acetate buffer (pH 3.9). The buffer for immobilizing S100β was 10 mM sodium acetate (pH 3.9). The amine coupling chemistry was applied for immobilization by using EDC/NHS to activate the CMDP surface and 1 M ethanolamine to deactivate non-reacted NHS-esters on the sensor surface.